The primary objective of this project is to define biochemical mechanisms involved in eukaryotic DNA synthesis. To approach this problem, we are investigating adeno-associated virus (AAV) and adenovirus DNA replication in in vitro systems. We have now shown that replicating forms of AAV DNA can be generated in vitro utilizing either endogenous or exogenously added templates and cellular polymerases. These studies also demonstrate that de novo initiation of DNA synthesis can occur in vitro. Partial fractionation of extracts has been accomplished, and two potent inhibitors of AAV DNA synthesis have been purified. Among methods used are differential centrifugation, ion exchange and affinity chromotography, gel electrophoresis and isoelectric focusing.